7s immunoglobulin for treatment of choroidal neovascularisation

ABSTRACT

The present invention refers to the use of 7 S immunoglobulin or an active fraction thereof for the manufacture of a medicament for the treatment of choroidal neovascularaisation and a pharmaceutical composition essentially consisting of an active fraction of 7 S immunoglobulin as pharmaceutically active compound and a method for the treatment of chorodial neovascularisation which comprises administering to the patient a preparation of 7 S immunoglobulin

TECHNICAL FIELD OF THE INVENTION

The present invention refers to the use of immunoglobulins for themanufacture of a medicament for the treatment of choroidalneovascularisation and a pharmaceutical composition and a method for thetreatment of choroidal neovascularisation.

BACKGROUND OF THE INVENTION

Choroidal neovascularisation is a desease wherein new capillaries andblood vessels are formed in uncontrolled manner in the choroid of theeye.

Subretinal neovascularisations of the eye (choroidal neovascularisations[CNV]) lead to rapidly declining vision and loss of quality of life. Atpresent the pathogenesis of the mechanism of CNV manifestation is notexactly known. The treatment of CNV is still a great ophthalmic-medicalproblem to be solved. Age-related macular degeneration is the leadingcause of legal blindness in people over 60 years of age in the WesternWorld (Kirchhof B., Deutsches Ärzteblatt 97 (21), (2000) A-1458-A-1462;Klaver et al., Arch. Ophthalmol. 116 (5) (1998) 653-658; Pauleikhoff,praktische Augenheilkunde 21 (2000) 59-65). The main reason for severeand progressive vision loss in AMD is the occurrence of subfoveal CNV(also termed wet or exsudative AMD) (Ferris, Arch. Ophthalmol. 102 (11)(1984) 1640-1642; Klaver et al., Arch. Ophthalmol. 116 (5) (1998)653-658). Diseases such as pathologic myopia, ocular histoplasmosissyndrome, traumatic incidents or even inadequate laser treatment cancause CNV in younger patients (Cohen et al., Ophthalmology 102 (8)(1996) 1241-1244; Derosa et al., Doc. Ophthalmol. 91 (3) (1995)207-222). Subretinal neovascular disease can also occur without anyobvious reason as idiopathic CNV (Cleasby, Am. J. Ophthalmol. 81 (5)(1976) 590-599). At present the pathogenesis of the mechanism of CNVmanifestation is not exactly known, although several angiogeneticfactors have been identified as potential risk factors (Campochiaro,Mol. Vis. 5 (1999) 34; D'Amato, Ophthalmology 102 (9) (1995) 1261-1262;Zarbin, Eur. J. Ophthalmol. 8 (4) (1998) 199-206).

The treatment of CNV is still a great ophthalmic-medical problem to besolved. The aim is to prevent the expected progression as shown in thenatural course of the disease. Improvement of the visual function isachieved only in a few cases.

State of Art:

Following treatments of choroidal neovascularisation have been discussedin the state of art:

Photodynamic Therapy

The primary mechanism of photodynamic therapy (PDT) is selective damageto fibrovascular choroidal tissue with preservation of overlying retinalstructures such as photoreceptors and retinal pigment epithelium. Theprocess of PDT involves the intravenous administration of alight-activated drug used as photosensitiser and its activation by aspecific wavelength of light using a non-thermal diode laser device(Schmidt-Erfurth, Ophtalmologe 95 (10) (1998) 725-731; Scott, DrugsAging 16 (2) (2000) 139-146). Verteporfin is the light-activated drugand active chemical component indicated for PDT (VIP Study Group 2001(a)Ophthalmology 108: 841-852; VIP Study Group 2001(b), Am. J. Ophthalmol.131: 541-560).

The risk of an increased photosensitivity reaction after Verteporfintherapy due to sunlight exposure is declared to last only for two days(CIBAVision (1999) Visudyne product information CIBA Vision AG.2-12-1999).

The efficacy of PDT in AMD patients with predominantly classic CNV hasbeen proven in long-term results of a controlled, randomised study bythe TAP Study Group (TAP Study Group (1999), Arch. Ophthalmol. 117 (10):1329-1345; TAP Study Group (2001) Arch. Ophthalmol. 119 (2): 198-207,Bressler, IOVS 41 (5) (2000) 624-628). The recommended efficacy resultsin these patients prescribed less than 3 respectively 6 lines of visualacuity loss compared with placebo, or preservation of a mean of 2 linesof visual acuity and 2 segments of contrast sensitivity, compared withplacebo.

Laser Treatment

As a result of the thermic effects of laser photocoagulation usingargon- or cryptonlaser localised damage of the retinal photoreceptorsand pigment epithelium is seen (Gloor, Schweiz. Rundsch. Med. Prax. 78(16) (1989) 467-470; Wu, Cur. Opin. Ophthalmol. 10 (3) (1999) 217-220).Laser photocoagulation is used successfully in diabetic patientspresenting with small and well-demarcated retinal mikrovascularabnormalities due to diabetes. In patients with subfoveal CNV only about25% of the patients meet eligible treatment criteria for lasercoagulation (MPS group, Arch. Ophthalmol. 109 (9) (1991) 1220-1241;Arch. Ophthalmol. 111 (9) (1993) 1220-1241; Arch. Ophthalmol. 112 (4)(1994) 489-499). The German Society of Ophthalmology published astatement on laser photocoagulation for subfoveal CNV saying that thismethod has not found world-wide acceptance for treatment of subfovealCNV (Gabel, _(“)Ärztliche Behandlung”. 13-7-2000).

Radiation Therapy

According to the German Society of Ophthalmology radiation therapy forAMD has not proved efficacy in controlled clinical studies (Gabel,_(“)Ärztliche Behandlung”. 13-7-2000). Several controlled studies showedno or insufficient effects on the visual function after radiationtherapy (RAD Study, Ophthalmology 12 (1999) 2239-2247; Anders et al.,Ophthalmologe 11 (1998) 760-764; Tholen, Ophthalmologe 95 (10) (1998)691-698). Furthermore the type or dose to be used for en effectiveradiation is not yet known. Therefore further controlled, randomisedstudies concerning radiation therapy in AMD are necessary (Archambeau etal., Int. J. Radiat. Oncol. Biol. Phys. 40 (5) (1998) 1125-1136; Berson,Semin. Radiat. Oncol. 9 (2) (1999) 155-162).

Pharmacological Therapy

No proven pharmacological treatment is known for subfoveal CNV. Neitherfor steroids (Challa, Aust. N. Z. J Ophthalmol 26 (4) (1998) 277-281),nor for the application of interferon alpha-2a (PTMD Study Group, Arch.Ophthalmol. 122 (5) (1997) 663-671) an efficacy could be proved. In AMDnew therapeutic approaches using Gingko biloba (Clostre, Ann. Pharm. Fr.57 Suppl 1 (1999) 18-88) and Phentoxyphyllin (Kruger et al., Arch.Ophthalmol. (1) (1998) 27-30) have been also tried.

New Approaches in Therapy of CNV

Further long-term results and controlled studies are needed to prove theefficacy of new therapeutic approaches in AMD such as submacularsurgery, retinal translocation and retina transplantation (Ciulla etal., Surv. Ophthalmol. 43 (2) (1998) 134-146; Joffe, Int. Ophthalmol.Clin. 36 (2) (1996) 99-116).

State of Art Regarding 7 S Immunoglobulin

Intravenous immunoglobulin (IVIg), also called 7 S immunoglobulin, isincreasingly used in the treatment of autoimmune and inflammatorydiseases, including vasculitides and Kawasaki disease.

Intravenous gammaglobulin (7 S immunoglobulin) has been in use since1981, primarily for prophylaxis in those with primary or secondaryimmunodeficiency states. Beneficial results have also been reported inthe treatment of childhood idiopathic thrombocytopenic purpura, in CMV(cytomegalie virus) prophylaxis for bone marrow transplantation,amelioration of GVHD (graft versus host disease), and other autoimmunedisorders. IVIg (7 S immunoglobulin) is known to contain antiidiotypicantibody activity against a number of autoantibodies (i.e. anti-ANA andanti-ANCA). IVIg (7 S immunoglobulin) is known to block antibodyresponse in vivo and in vitro although the exact mechanisms are notknown. IVIg (7 S immunoglobulin) has been used successfully to obtainimproved post transfusion platelet-increments in refractory patients.

Intravenous immunoglobulin (7 S immunoglobulin) is therapeuticimmunoglobulin (Ig) prepared from pools of plasma of several thousandhealthy blood donors. In addition to its use as substitutive therapy forprimary and secondary antibody deficiencies, IVIg (7 S immunoglobulin)exhibits immunomodulatory effects in diseases mediated by autoantibodiesand in diseases believed to be primarily mediated by autoaggressive Tcells in humans and in experimental animals. IVIg (7 S immunoglobulin)has been used effectively in the treatment of autoimmune cytopenias, theacute Guillain-Barré-syndrome, myasthenia gravis and anti-factor VIIIautoimmune disease. Patients suffering from systemic inflammatoryconditions such as dermatomyositis and, particularly Kawasaki syndromegreatly benefit from IVIg treatment. IVIg (7 S immunoglobulin) has alsobeen used in the treatment of anti-neutrophil cytoplasmicantigen-associated systemic vasculitis. The mechanisms of action if IVIgare, as yet, poorly understood, although several mutually nonexclusivehypotheses have been proposed. These include the blockade of Fc γreceptors on phagocytic cells, interference with activated complementmodulation of production and release of cytokines and their inhibitors,modulation of T- and B-lymphocyte functions, suppression of autoantibodyproduction, and selection of immune repertoires.

U.S. Pat. No. 5,562,902 discloses a therapeutic method for inhibitingtumour metastasis and treatment of primary tumours comprisingadministering to a patient a preparation of intravenous gammaglobulin(IVIg).

U.S. Pat. No. 6,171,585 refers to methods of transplantation and tomethods to immunosuppress a potential transplant recipient so as to beamenable to transplant with donor organs obtained from a variety ofdonors including histoincompatible donors. The method for transplantingan allograft in a patient comprises administering to the patient priorto transplantation an effective amount of an anti-HLA-antibody depletingagent which is essentially intravenous immunoglobulin.

The German patent application DE 199 00 503 A1 discloses the use of acomposition for the manufacture of a medicament for the treatment ofepidermal necrolysis, graft-versus-host disease, hepatitis, autoimmunethyroiditis, cancer or HIV.

On the cellular level the induction of neovascularisation, i.e. theformation of new blood vessels capillaries, may be explained as follows:The two major cellular components of the vasculature are the endothelialand smooth muscle cells. The endothelial cells form the lining of theinner surface of all blood vessels, and constitute a nonthrombogenicinterface between blood and tissue. In addition, endothelial cells arean important component for the development of new capillaries and bloodvessels. Thus endothelial cells proliferate during the angiogenesis, orneovascularization, associated with tumor growth and metastasis, as wellas a variety of non-neoplastic diseases or disorders. Duringangiogenesis, or neovascularization endothelial cells proliferate,migrate and are responsible for tube formation of the future bloodvessel or capillary.

The vascular endothelium is strategically located between thecirculating blood and the vascular smooth muscle cells. Differentagonists or stimuli transported by the circulating blood can trigger theendothelium to release potent relaxing (nitric oxide, prostacyclin,endothelium-derived hyperpolarizing factor) or contracting factors(endothelin, cycloxygenase products). These endothelium-derivedvasoactive factors can modulate blood flow locally (Haefliger et al.;Prog. Retin. Eye Res. 20, (2001) 209-225).

Heterogeneity exists from one vascular bed to the other, or even betweenvessels, in the agonists able to stimulate the release ofendothelium-derived vasoactive factors. In the ophthalmic circulation,nitric oxide and endothelin are strong vasoactive modulators. In manyvascular diseases that are of importance in ophthalmology(hypercholesterolemia, arteriosclerosis, hypertension, diabetes,vasospastic syndrome, ischemia and reperfusion, choroidal and retinalneovascularisation, age related macular degeneration, diabeticretinopathy) the function of the endothelium can be impaired.Endothelial cells (ECs) are very heterogeneous cells that differ byacquisition and maintenance of specialized properties which is importantfor the functional homeostasis of different organs (Garlanda C, DejanaE: “Heterongenity of endothelial cells. Specific markers.” InArterioscler. Thromb. Vasc. Biol. 17, (1997) pp 1193-1202).

Endothelial cells also have site-specific differences in the sensitivityto cell injury (Murphy et al. Heterogeneity of vascular endothelialcells: differences in susceptibility to neutrophil-mediated injury;Microvasc. Res. 56, (1998), pp 203-211). Especially in the eye,alteration of the blood-retina barrier have important consequences oneye functional integrity. Even in the eye ECs and blood vessels fromretina and choroid differ largely. For instance in the rat, choroidalarterioles are much larger in diameter than retinal arterioles which mayexplain differences of hemodynamics of both tissues (Ninomiya H, Kuno H;Vet. Ophthalmol. 4, (2001) pp 55-59). In addition ECs from thechoriocapillaris are fenestrated whereas retinal ECs are not. Thefenestrated capillaries in the choroid are very permeable to lowmolecular weight substances; sodium permeability in the choroid isprobably 50 times that in skeletal muscle (Tornquist P, Alm A, Bill A;Eye 4 (Pt 2), (1990) pp 303-309). These results in high concentrationsand rapid turnover of nutrients in the extra-vascular compartment of thechoroid. Also the retinal capillaries, with tight junctions between theendothelial cells, have very low permeability even to sodium in contrastto the choriocapillaris.

Biochemical variations, such as the expression of ion channels, connexinsubtypes and other important components of second messenger cascades,have been documented in the smooth muscle and endothelial cells indifferent parts of the body (Hill C E, Phillips J K, Sandow S L (2001):Med. Res. Rev. 21 (2001), 1-60). Anatomical variations, in the presenceand prevalence of gap junctions between smooth muscle cells, betweenendothelial cells and at myoendothelial gap junctions, between the twocell layers, have also been described. These factors will contributefurther to the heterogeneity in local and conducted responses (Hill C E,Phillips J K, Sandow S L; Med. Res. Rev. 21, (2001), pp 1-60).

There exist different drugs that can modulate the vasoactive function ofthe vascular endothelium. In other words, it appears that the vascularendothelium plays an important role in both the physiology andpathophysiology of the regulation of blood flow. The modulation of thisregulatory system by different drugs might open new therapeuticalapproaches to treat vascular disorders in ophthalmology. It has beenshown that immunoglobulin can inhibit one parameter ofneovascularization, which is proliferation in human umbilical veinendothelial cells in vitro (Xu C. et al.: Modulation of endothelial cellfunction by normal polyspecific human intravenous immunoglobulines. Am.J. Pathol. 153, (1998), 1257-1266). Xu et al. describes that by usinghuman umbilical vein endothelial cells (HUVECs) as target cells, IVIg (7S immunoglobulin) from different commercial sources modulates thefunction of endothelial cells (ECs). In their studies IVIg (7 Simmunoglobulin) inhibited EC proliferation in a dose- and time-dependentmanner. It was also shown that IVIg down-regulated the TNF-α orIL-1β-induced expression of mRNA encoding major adhesion molecules,chemokines, and proinflammatory cytokines, which are significantlyimplicated in the leukocyte recruitment observed in several inflammatorydiseases. However, these cells (human umbilical vein endothelial cells)are not an ideal model, since they are close to senescence and arecultured from hypoxic and possibly activated vessels (Garlanda C, DejanaE; Arterioscler. Thromb. Vasc. Biol. 17, (1997), pp 1193-1202).Therefore, it is preferable to culture the endothelium from themicrovasculature of the target organ and to maintain their specializedproperties in vitro.

Pathological neovascularization is not only characterized byproliferation but also by migration and tube formation of endothelialcells. The effect on migration and tube formation of endothelial cellsby immunoglobulin has not yet been shown. The effects of immunoglobulinon endothelial cells from the eye are completely unknown.

In view of the state of art, there did exist a need agents showinginhibitory activities on choroidal endothelial cells and choroidalneovascularisation. There also exist a need for medicaments and methodsfor the treatment of choroidal neovascularisation (CNV).

Therefore it was technical object of the present invention to provide amedicament for the treatment of choroidal neovascularisation.

SUMMARY OF THE INVENTION

The present invention generally solves the problems referred to above byproviding the use of 7 S immunoglobulin or an active fraction thereoffor the manufacture of a medicament for the treatment of choroidalneovascularisation.

In particular, it is provided the use of 7 S immunoglobulin or an activefraction thereof for the manufacture of a medicament for the treatmentof choroidal neovascularisation, said 7 S immunoglobulin or said activefraction thereof having the ability to inhibit one or more activitiesselected from the group consisting of:

a) choroidal neovascularisation,

b) proliferation of choroidal endothelial cells,

c) migration of choroidal endothelial cells,

d) tube formation of choroidal endothelial cells.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein “gamma globulin” or “7 S immunoglobulin” is the serumglobulin fraction that is mainly composed of IgG molecules.

As used herein, “IVIg” or “intravenous immunoglobulins” or “7 Simmunoglobulin” refers to a gamma immunoglobulin fraction which may beprepared by fractional alcohol precipitation (such as according toCohn-Oncley-method) from human blood plasma, such as those 7 S gammaimmunoglobulin preparations commercially available from several sources.The 7 S immunoglobulin fraction again essentially consists of theimmunoglobulins IgG₁, IgG₂, IgG₃ and IgG₄.

As used herein, _(“)active” fraction of 7 S immunoglobulin refers topharmaceutically activity in terms of being effective in the treatmentof choroidal neovascularisation. The term “active” does also refer to abiological (and pharmaceutically) activity as being effective ininhibiting choroidal neovascularisation, inhibiting proliferation ofchoroidal endothelial cells, inhibiting migration of choroidalendothelial cells, inhibiting tube formation of choroidal endothelialcells.

Intravenous immunoglobulins (7 S immunoglobulin) are therapeuticpreparations of normal polyspecific IgG obtained from plasma pools ofover 6,000 healthy blood donors. Currently used preparations are made ofintact IgG with a distribution of subclasses corresponding to that ofnormal serum and have a half-life of three weeks in vivo for IgG₁, IgG₂and IgG₄, and somewhat less for IgG₃. Most of the preparations containonly traces of IgA, IgM and of Fc-dependent IgG aggregates (see Kaveriet al., in Clin. Exp. Immunol. 86 (1991) 192-198).

As used herein “fragments” of IVIg or gamma globulin or 7 Simmunoglubulin are portions of intact immunoglobulins such as Fc, Fab,Fab′, F(ab′)₂ and single chain immunoglobulins.

7 S immunoglubulin preparations contain up to 30% (w/w) ofF(ab′)₂-F(ab′)₂ dimers as assessed by size-exclusion chromatography andelectronmicroscopy. The dimers are the consequence of V-regioncomplementarity between immunoglobulins in the pool (see Roux &Tankersley, in J. Immunol. 134 (1990) 1387). Owing to the large numberof donors, IVIg (7 S immunoglobulin) represent a wide spectrum of theexpressed normal human IgG repertoire, including antibodies to externalantigens, autoreactive antibodies and anti-antibodies.

Commercial IVIg (7 S immunoglobulin) preparations are widely available,for example, from Aventis Behring, Cutter Laboratories, MedImmune;Novartis Pharma (Nürnberg, Germany), Octapharma, Venoglobulin, MilesInc. (West Haven, Conn.), N.V. Baxter S.A. (Lessines, Belgium), SandozPharma Ltd. (Basle, Switzerland), Instituto Sierovaccinogeno Italiano(Isiven, Italy) and Jackson Immunoresearch Laboratories, Inc. (WestGrove, Pa.). The commercially available IVIg (7 S immunoglobulin)preparations contain mainly IgG molecules and in maltose or glycinecarriers. Also contemplated for use herein are aqueous solutionscontaining higher concentrations of IVIg (7 S immunoglobulin), such asapproximately 25% (w/w)-75% (w/w). Substantially pure preparations ofthe “IgG-fraction of IVIg” are also suitable for use herein.Substantially pure IgG-fractions typically contain greater than 50%(w/w) of an IgG-fraction, preferable greater than 75% (w/w), and mostpreferably greater than 95% (w/w) of an IgG-fraction. Such substantiallypure IgG-fractions are commercially available from several sources.

According to a National Institutes of Health (NIH) Consensus Conferencereport, the incidence of adverse side effects associated with IVIg usein humans, used at dosage regimens comparable to the ones contemplatedby the present invention, is usually less than 5% with most of thosereactions being “mild and self-limited”. The report adds that “severereactions occur very infrequently and usually do not contraindicatefurther IVIg therapy”. the NIH report also notes that “[n]either HIV norhepatitis B infection has been transmitted to recipients of productscurrently licensed in the United States”. NIH Consensus Conference,“Intravenous Immunoglobulin: Prevention and Treatment of Disease”, JAMA,264, pp. 3189-3193 (1990).

The 7 S immunoglubulin preparations that may be used according to thepresent invention include commercially available preparations of intact7 S immunoglubulin and preparations of the F(ab′)₂ fragments of 7 Simmunoglubulin. Recombinantly produced gamma globulin and theirfragments may also be used according to this invention. The use ofrecombinant single chain antibodies is also envisioned.

The dosage of 7 S immunoglubulin and the method of administration willvary with the severity of the particular condition being treated, theduration of treatment, the adjunct therapy used, the age and physicalcondition of the subject of treatment and like factors within thespecific knowledge and expertise of the treating physician. However,singe dosages for intravenous and intracavitary administration cantypically range from 400 mg to 2 g per kilogram body weight, preferably2 g/kg (unless otherwise indicated, the unit designated “mg/kg” or“g/kg”, as used herein, refers to milligrams or grams per kilogram ofbody weight). The preferred dosage regimen is 400 mg/kg/day for 5consecutive days per month or 2 g/kg/day once a month. According to thepresent invention 7 S immunoglubulin was found to be effective in thetreatment of choroidal neovascularisation when administered byintravenous or intraperitoneal injection and in the dose range of500-1000 mg/kg/week.

In an other embodiment of this invention, the 7 S immunoglubulinpreparation is administered via the subcutaneous route. The typicaldosage for subcutaneous administration can range from 4 mg to 20 mg perkg body weight. According to the present inventions 7 S immunoglubulinwas found to be effective in the treatment of choroidalneovascularisation administered subcutaneously in the dose 500-1000mg/kg/week.

According to the present invention 7 S immunoglubulin may beadministered as a pharmaceutical composition containing apharmaceutically acceptable carrier. The carrier must be physiologicallytolerable and must be compatible with the active ingredient. Suitablecarriers include sterile water, saline, dextrose, glycerol and the like.In addition, the compositions may contain minor amounts of stabilisingor pH buffering agents and the like. The compositions are conventionallyadministered through parenteral routes, with intravenous intracavitaryor subcutaneous injection, being preferred.

Surprisingly it has been found that intravenous 7 S immunoglubulin iseffective in the treatment of choroidal neovasularisation (FIGS. 4 to8). Administration of intravenous 7 S immunoglubulin resulted in asignificant improvement of visual acuity.

The inventors of the present invention carried out studies by using 7 Simmunoglobulines for treatment of CNV in young patients (diagnosis e.g.idiopathic CNV, presumed ocular histoplasmosis syndrome [POHS]) and inolder patients (diagnosis e.g. age-related macular degeneration [AMD]).The treatment was performed in repeated therapeutic cycles and 7 Simmunoglobulin was given intravenously. The efficacy results have beenproved by the change of the visual acuity with ETDRS-charts (EarlyTreatment Diabetic Retinopathy Study, Lighthouse, N.Y.). The change ofthe visual acuity is measured in lines on the EDTRS-chart, whereby achange of one line corresponds to a change of 0.1 LogMAR units. TheETDRS-charts have been developed especially for patients withmaculopathy (Ferris et al. 1982).

Intravenous 7 S immunoglubulin does also inhibit the proliferation(FIGS. 1 and 2), migration and tube formation of choroidal endothelialcells in cell culture.

Although Xu et al. (Am. J. Pathol. 153 (4) (1998), pp. 1257-1266) havefound that IVIg inhibited proliferation of endothelial cells, theseresults could not be transferred to the choroidal endothelial cells. Xuet al. did show this effect on human umbilical vein endothelial cells(HUVECs). However, it is known in the art that endothelial cells ofdifferent tissues show a strong heterogeneity.

Garland C. and Dejana E. summarise the observations made on theheterogeneity of endothelial cells in their review article“Heterogeneity of Endothelial Cells” in Arteriosclerosis, Thrombosis andVascular Biology (1997; 17:1193-1202). In their review article theauthors describe that the endothelium is considered a sparse organsystem, due to its vast extension and ability to exert a complex arrayof specialised functions. A unique characteristic of endothelial cells(ECs) is that, although they present many common functional andmorphological features, they also display remarkable heterogeneity indifferent organs. Even in the same organ, the endothelium of large andsmall vessel, veins and arteries exhibits significant heterogeneity. Anextreme case is the kidney, which contains different types of ECs:fenestrated in the peritubular capillaries, discontinuous in glomerularcapillaries and continous in other regions. Embryonic ECs seemparticularly “plastic.” Most of the specialised characteristics of ECsare induced during development, whereas adult endothelium is not equallysusceptible to differentiation factors. Despite its stable constitutiveproperties, the adult endothelium can reversibly change its functions onactivation. Adult ECs can be reprogrammed according to the transitoryneeds of the organism. For instance, exposure of ECs to inflammatorycyokines, such as IL-1 and tumour necrosis factor, or to growth factors,such as VEGF or FGF, induces a complex functional reprogramming, whichimplies the neosynthesis of some genes and the repression of others. ECscan be activated several times during their life span by the same ofdifferent cytokines and thereby display different and reversiblephenotypes.

As mentioned above endothelial cells (ECs) display remarkableheterogeneity in different organs and even in the same organ, theendothelium of large and small vessel, veins and arteries exhibitssignificant heterogeneity. The inventors of the present inventionperformed studies with human umbilical vein endothelical cells (HUVECs),retinal endothelical cells and choroidal endothelial cells. 7 Simmunoglobulin did show inhibitory effects on choroidal endothelialcells and HUVECs. However, it did not show any effects on retinalendothelial cells at all (see FIG. 3). Since endothelial cells from thesame organ (from retina and choroid, respectively) show such extremelydifferent sensitivity to 7 S immunoglobulin the person skilled in theart would not have expected that 7 S immunoglobulin could be used as amedicament for the treatment of choroidal neovascularisation.

In a preferred embodiment said active fraction is selected from thegroup consisting of IgG fractions of 7 S immunoglobulin IgG₁, IgG₂,IgG₃, IgG₄ and mixtures thereof.

The distribution of the subclasses of 7 S immunoglobulin in themedicament may correspond to the one in the blood serum of a healthyperson. Preferably the distribution of the IgG subclasses in themedicament is as follows:

-   IgG₁: 60-70% (w/w)-   IgG₂: 25-30% (w/w)-   IgG₃: 3-8% (w/w)-   IgG₄: 0.5-3% (w/w).

In a further preferred embodiment said 7 S immunoglobulin is present inthe medicament as a composition comprising at least 50% (w/w),preferably at least 80% (w/w), more preferred at least 95% (w/w) gammaimmunoglobulin.

In a further preferred embodiment said active fraction of 7 Simmunoglobulin is present in the medicament in an amount of at least 50%(w/w), preferably of at least 80% (w/w), more preferred of at least 95%(w/w).

Preferaby said active fraction is composed of fragments of gammaimmunoglobulin. The medicament which is used for the treatment ofchoroidal neovascularisation may contain fragments of gammaimmunoglobulin which are selected from the group consisting of F(ab′)₂,Fab′, Fab and Fc of gamma immunoglobulin.

In yet another embodiment the medicament will contain gammaimmunoglobulin in an amount effective to inhibit choroidalneovascularisation. Particularly, the gamma immunoglobulin will bepresent in the medicament in an amount effective to inhibit the growthof new blood vessels in the eye. In a further preferred embodiment thegamma immunoglobulin is present in the medicament in an amount effectiveto inhibit the proliferation of choroidal endothelial cells. In afurther preferred embodiment the gamma immunoglobulin is present in themedicament in an amount effective to inhibit migration of choroidalendothelial cells and effective to inhibit tube formation which isinitiated by choroidal endothelial cells when new blood vessels develop.

The present invention also solves the problems referred to above byproviding a Pharmaceutical composition essentially consisting of anactive fraction of 7 S immunoglobulin as pharmaceutically activecompound.

Preferably, said active fraction in the pharmaceutical composition hasthe ability to inhibit one or more activities selected from the groupconsisting of:

a) choroidal neovascularisation,

b) proliferation of choroidal endothelial cells,

c) migration of choroidal endothelial cells,

d) tube formation of choroidal endothelial cells.

In a further preferred embodiment said active fraction is one, two orthree of the components selected from the group consisting of IgGfractions of 7 S immunoglobulin IgG₁, IgG₂, IgG₃, IgG₄.

In another preferred embodiment said active fraction of 7 Simmunoglobulin is present in the pharmaceutical composition in an amountof at least 50% (w/w), preferably of at least 80% (w/w), more preferredof at least 95% (w/w).

The active fraction preferably is composed of fragments of gammaimmunoglobulin, more preferred the fragments of gamma immunoglobulin areselected from the group consisting of F(ab′)₂, Fab′, Fab and Fc of gammaimmunoglobulin.

The active fraction contained in the pharmaceutical composition of thepresent invention is contained in an amount effective to inhibitchoroidal neovascularisation and growth of new blood vessels in the eye.Furthermore, the active fraction is contained in an amount effective toinhibit the proliferation of choroidal endothelial cells, the migrationof choroidal endothelial cells, and the tube formation initiated bychoroidal endothelial cells.

The present invention also solves the problems referred to above byproviding therapeutic methods for the treatment of choroidalneovascularisation which comprises administering to the patient apreparation of 7 S immunoglobulin or an active fraction thereof.

Preferably, said 7 S immunoglobulin or said active fraction of 7 Simmunoglobulin is administered in an amount effective to inhibitchoroidal neovascularisation. In a further preferred method for thetreatment of choroidal neovascularisation, said 7 S immunoglobulin or anactive fraction thereof have the ability to inhibit one or moreactivities selected from the group consisting of:

a) choroidal neovascularisation,

b) proliferation of choroidal endothelial cells,

c) migration of choroidal endothelial cells,

d) tube formation of choroidal endothelial cells.

Further embodiments of the method of the present invention comprisefeatures mentioned above in reference to the use of 7 S immunoglobulinfor the manufacture of the medicament for the treatment of choroidalneovascularisation.

The present invention will be explained in more detail by the followingexamples and with reference to the FIGS. 1 to 8, which are not to beconstrued as limiting the scope of the present invention in any manner.

BRIEF DESCRIPTION OF THE FIGURES

Figure Legends

FIG. 1 shows the inhibition of choroidal endothelial cell proliferationby IgG (7 S immunoglobulin) from 2 independent experiments after 48hours. Proliferation is inhibited in a concentration dependent manner.This experiment was performed to find the range of efficient dosage.

FIG. 2 shows the inhibition of choroidal endothelial cell proliferationby IgG (7 S immunoglobulin) from 4 independent experiments, that aredifferent from the experiment presented in FIG. 1, after 48 hours incomparison to an untreated control. Proliferation is inhibited in aconcentration dependent manner.

FIG. 3 shows the effect of 40 mg/ml IgG (7 S immunoglobulin) on ECmigration after 8 hours depending on the cells origin. Migration isinhibited in choroidal ECs by 56% and by 35% in HUVECs. The inhibitionof IgG is absent in retinal ECs.

FIG. 4 shows the change of visual acuity in 10 patients with idiopathicCNV or POHS over a period of 18 months after treatment with 7 Simmunoglobulin. After 12 months 9/10 eyes and after 18 months 8/10 eyesimproved in visual acuity.

FIG. 5 shows the change of visual acuity of these eyes after 3 and 18months presented as box-plots with the corresponding median aftertreatment with 7 S immunoglobulin. The improvement is significant.

FIG. 6 shows the change of visual acuity in a young male patient withidiopathic CNV in the right eye (OD). The period of time of treatmentwith 7 S immunoglobulin is marked by an arrow (Treatment).

FIG. 7 shows the change of visual acuity in a young female patient withPOHS in both eyes (right eye: OD, left eye: OS). The period of time oftreatment with 7 S immunoglobulin is marked by an arrow (Treatment).

FIG. 8 shows the change of visual acuity in a 70 year old patient withCNV caused by age-related macular degeneration (AMD) after treatmentwith 7 S immunoglobulin. The stars mark the change of visual acuitybefore a therapeutic cycle. Before treatment the visual acuity on thebetter right eye (OD) was 0.125.

EXAMPLES

Methods

Preparation of Primary Human Choroidal or Retinal Endothelial Cells

Human eyes were obtained within 30 h of death from the donors. They werefree of known ocular diseases. The eyes were dissected, and the anteriorsegment, vitreous and retina were separated. The choroid with retinalpigment epithelium layer or the retinas were incubated for 30 min at 37°C. with Accutase (PAA). Then the retinal pigment epithelium cells wereremoved with a spatula. The choroid was stripped off the sclera, washedwith Hanks' balanced salt solution (HBSS) and then incubated with 0.25%trypsin and 0.02% EDTA at 37° C. for 1 h. The choroidal or retinalfragments were further incubated with HBSS containing collagenase 4000U/ml (1:4 in HBSS) for 30 min at 37° C., washed twice with HBSS andfurther incubated with collagenase for 2 h at 37° C. Then the choroidalor retinal fragments were passed through sterile mesh (70 μm pore size),the suspension centrifuged (5 min at 1500 rpm) and the supernatantdiscarded. The pellet was washed with HBSS containing 1% BSA and onceagain centrifuged. The cell pellet was resuspend in 1 ml HBSS (1% BSA)and transferred to 1.5 ml eppendorf tubes. For separation of theendothelial cells magnetic beads (Dynabeads CD-31, dynal, Cat. N 111.28)precoated with a IgG1 monoclonal antibody (clone 9G11) specific for thehuman CD31 cell surface antigen were used. The magnetic beads werewashed several times with 1% BSA in HBSS and then 10 μl of the beadssolution (1×107 beads per 1 ml cell suspension) were added to the cellsuspension and mixed gently for 2 h at room temperature. After that, thetubes were placed on Dynal Magnetic particle Concentrator (Dynal MPC®)and allowed to sediment for 2 min. The supernatants were removed and thecells attached to the magnetic beads were washed with 1% BSA in HBSS,resuspended in endothelial cell growth medium (Promocell, cat. N 22020)containing 0.4% ECGS/H, 5% FCS, 10 ng/ml EGF, 1 ng/ml hydrocortison, 50ng/ml bFGF, 50 ng/ml Amphotericin B, 50 μg/ml Gentamicin and seeded onculture dishes.

Human umbilical vein endothelial cells (HUVECs) were bought fromPromoCell (Heidelberg, Germany).

Immunocytochemistry

The purity of the cell preparation was determined by endothelial cellspecific antibodies. For immunocytochemistry, the cells were seeded onimmunochambres at a density of 5×10³ cells/chamber and cultured for 5days in endothelial cell growth medium. After fixation with 4%paraformaldehyde at 4° for 20 min, cells were washed three times withphosphate buffered saline (PBS). To block the unspecific bindings, thecells were incubated for 1 h with 5% BSA in PBS. After washing, thecells were reacted with anti-human Von Willebrand factor antigen rabbitpolyclonal, dilution 1:200 in PBS, with 5% rabbit serum, Dako, Cat. NA0082) overnight at 4° C. The cells were washed five times with PBS andthen incubated with a secondary antibody conjugated with alkalinephosphatase (anti-rabbit IgG, dilution 1:400 in PBS, 1% sheep serum,Sigma) for 2-3 h at room temperature, washed four times with PBS andexposed to new fuchsin for 5 min. Hemalun contrast staining wasperformed. Then the cells were washed and mounted with KaisersGlyceringelatine and observed by light microscopy. As a negativecontrol, normal rabbit serum (diluted 1:700 in PBS) was used instead ofthe primary antibody.

Statistical Analysis:

For statistical analysis Student's t-test was used.

Example 1: Cell Proliferation Assay

Choroidal endothelial cells were seeded on 96-well culture plates (Nunc)at a density of 1000 cells/well and cultured in endothelial cell growthmedium (5% FCS) for 24 h. The medium was discarded, the cells werewashed three times with PBS and exposed to basal endothelial cell mediumonly (control) containing 50 ng/ml VEGF (positive control). The cellswere exposed to 5 mg, 10 mg, 20 mg and 40 mg immunoglobulin,respectively (Sandoglobulin®, Novartis Pharma, Nürnberg, Germany).

WST-1 assay was used to determine the proliferation rate of endothelialcells according to the manufacturer's instructions (BoehringerMannheim). The colorimetric proliferation assay is based on the cleavageof the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viablecells. The most effective dose of VEGF was determined by theproliferation assay in a pilot study on days 1, 3, and 5 after platingthe endothelial cells.

Results:

The stimulatory effect of VEGF (50 ng/ml) on CEC proliferation wassignificantly blocked after 48 hours in a concentration dependent mannerby exposure of the cells to 20 or 40 mg IG/ml (n=4) by 33% (p=0.004) or45% (p=0.00004). The results are shown in FIGS. 1 and 2.

Example 2: Migration Assay

Migration of choroidal, retinal and umbilical vein ECs was assayed usingFluoroBlock Inserts according to the manufacturer instructions (Falcon).The assay is based on light-tight polyethylene terephthalate (PET)microporous membrane (8 μm pore size) which are constructed tospecifically detect fluorescence of labeled cells and molecules belowthe insert. The membranes were coated with gelatin (0.1% in 0.1 M PBS)for 1 h at a 37° C. Then the inserts were exposed to 1) medium+50 ng/mlVEGF (positive control); 2) medium+50 ng/ml VEGF containing 40 mg/mlImmunoglobulin; ECs (passages 2 to 4) were serum-starved (DMEM, 0.5%FCS) for 3 h, collected with 0.02% EDTA and loaded into the membranes ofinserts (5×10³ cells/per membrane). After 8 h of incubation at 37° C.,the filters were removed, washed with 0.1% PBS and fixed in 4%paraformaldehyde (in 0.1% PBS) for 30 min at room temperature. Thefilters were washed again with PBS and the cell nuclei were stained with4′,6-diamidino-2-phenylindole (DAPI) producing a blue fluorescence (460nm). Further, the filters were mounted with Vectashield Mounting medium(Vector Lab) bottom side up on glass slide. The numbers of cells inthree different microscopic fields (×10 objective) were counted. The netnumber of migrated cells was obtained by subtracting the number ofmigrated cells in the absence of chemoatractant from that in thepresence of such a stimulant.

Results:

The effect-of IG on ECs migration after 8 hours was different anddepended on the cells origin. Migration was inhibited in choroidal ECsby 56% and by 35% in HUVECs. The inhibition by IG was absent in retinalECs (FIG. 3).

Example 3: In Vitro Tube-Formation Assay

Growth factor depleted Matrigel (Harbor extracellular matrix basementmembrane), Tebu, cat. N 2001) was applied into a 24-well tissue cultureplate (400 μl/well) and forms an in vitro basement membrane. Afterpolymerization of the Matrigel (37° C., 1 h), primary human choroidalendothelial cells were seeded on the top of the gels at a density of 10000 cells/well and cultivated in endothelial cell growth mediumcontaining 5% FCS for 24 h. Then the medium was aspirated and the cellswere exposed to: 1) basal endothelial cell medium (contains no growthfactors, 2% FCS, Promocell cat. N 22210) (control); 2) basal endothelialcell medium containing 50 ng/ml VEGF; 3) conditioned medium (CM)(keratinocyte SF, Gibco Cat. N) from Ad.PEDF-infected primary rat IPEcells; 4) CM from AD.PEDF-infected rat IPE cells+50 ng/ml VEGF; 5) CMfrom Ad.eGFP-infected rat IPE cells; 6) CM from Ad.eGFP-infected rat IPEcells+50 ng/ml VEGF; 7) CM from non-infected rat IPE cells. The platewas incubated at 37° C. for 24 h, and then the medium was aspirated andcells were fixed in neutral buffered 10% formalin. The gel proteinsallow cell alignment and tube formation, which can be seen under aninverted light microscope. Representative pictures were taken at ×10magnification.

Results:

Tube formation by CEC was apparent in all experimental groups but wasless prominent in the presence of 40 mg/ml IG.

Conclusion:

Blockade of cellular proliferation, migration and tube formation mayexplain and confirm the therapeutic effect by 7 S immunoglobuline onchoroidal neovascularisation described below in example 4.

Example 4: Treatment of Patients Suffering from CNV

Studies were carried out by using 7 S immunoglobulines for treatment ofCNV in young patients (diagnosis e.g. idiopathic CNV, presumed ocularhistoplasmosis syndrome [POHS]) and in older patients (diagnosis e.g.age-related macular degeneration [AMD]).

The treatment was performed in therapeutic cycles over a period of 3-5days, which were repeated according to the clinical course. A totalamount of 50-100 g 7 S immunoglobulines were given intravenously in atherapeutic cycle. The heart and circulation parameters were surveyedduring and after the infusion. The efficacy results have been proved bythe change of the visual acuity with ETDRS-charts (Early TreatmentDiabetic Retinopathy Study, Lighthouse, N.Y.). The change of the visualacuity is measured in lines on the EDTRS-chart, whereby a change of oneline corresponds to a change of 0.1 LogMAR units. The ETDRS-charts havebeen developed especially for patients with maculopathy (Ferris et al.New visual acuity charts for clinical research; Am. J. Ophthalmol.; 94(1982) pp 91-96). Besides the clinical evaluation of the retinafundusphotographs and fluorescein-angiographies were performed fordocumentation.

10 younger patients with an idiopathic CNV or POHS were treated over amean period of 18 month by repetitive infusion of intravenous 7 Simmunoglobulins. The mean age of the patients was 38.5 years. Thetreatment was applied every 3 to 6 weeks at an individual base. A totaldose of 50 to 100 g was administered during each treatment cycle.Clinical endpoint of the study was visual acuity measured by ETDRScharts. The results are summarized in FIGS. 4 to 8.

After the trial 9 out of 10 patients showed an improvement of visualacuity. The mean improvement was 2.3 lines (p<0.01) after three and 3.2lines (p=0.02) after 18 months. According to the fluorescein angiographya partial involution and scaring of the CNV was observed. The studydemonstrated the beneficial effect of intravenous 7 S immunoglobulins onthe natural course of idiopathic CNV. The repetitive treatment led tofunctional and morphological improvements. The results are summarized inFIG. 5.

As example for an older patient FIG. 8 summarizes the results of thetreatment by showing the change of visual acuity in a 70 year oldpatient with CNV caused by age-related macular degeneration (AMD).

The treatment was tolerated well in general. The ophthalmologic clinicalcourse showed generally a reduction of exsudation, a reduction ofprogression of CNV and transition into a non-active stadium of scarring.

Conclusion:

The clinical results show a positive effect of a treatment with 7 Simmunoglobulines on the natural course of CNV.

1-79. (canceled)
 80. Use of 7 S immunoglobulin or gamma globulin or aserum globulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin for the manufacture of a medicament for thetreatment of choroidal neovascularisation.
 81. Use of an active fractionof 7 S immunoglobulin or gamma globulin or a serum globulin fractionthat is mainly composed of IgG molecules or intravenous immunoglobulinfor the manufacture of a medicament for the treatment of choroidalneovascularisation.
 82. Use of 7 S immunoglobulin or gamma globulin or aserum globulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin for the manufacture of a medicament for thetreatment of choroidal neovascularisation, said 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin having the ability toinhibit one or more activities selected from the group consisting of: a)choroidal neovascularisation, b) proliferation of choroidal endothelialcells, c) migration of choroidal endothelial cells, d) tube formation ofchoroidal endothelial cells.
 83. Use of an active fraction of 7 Simmunoglobulin or gamma globulin or a serum globulin fraction that ismainly composed of IgG molecules or intravenous immunoglobulin for themanufacture of a medicament for the treatment of choroidalneovascularisation, said active fraction having the ability to inhibitone or more activities selected from the group consisting of: a)choroidal neovascularisation, b) proliferation of choroidal endothelialcells, c) migration of choroidal endothelial cells, d) tube formation ofchoroidal endothelial cells.
 84. Use of 7 S immunoglobulin or gammaglobulin or a serum globulin fraction that is mainly composed of IgGmolecules or intravenous immunoglobulin for the manufacture of amedicament for the treatment of choroidal neovascularisation in anamount effective to inhibit choroidal neovascularisation.
 85. Use of anactive fraction of 7 S immunoglobulin or gamma globulin or a serumglobulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin for the manufacture of a medicament for thetreatment of choroidal neovascularisation, wherein said active fractionis present in the medicament in an amount effective to inhibit choroidalneovascularisation.
 86. Use of 7 S immunoglobulin or gamma globulin or aserum globulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin for the manufacture of a medicament for thetreatment of choroidal neovascularisation, said 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin having the ability toinhibit one or more activities selected from the group consisting of: a)choroidal neovascularisation, b) proliferation of choroidal endothelialcells, c) migration of choroidal endothelial cells, d) tube formation ofchoroidal endothelial cells, wherein said 7 S immunoglobulin or gammaglobulin or a serum globulin fraction that is mainly composed of IgGmolecules or intravenous immunoglobulin is present in the medicament inan amount effective to inhibit choroidal neovascularisation.
 87. Use ofan active fraction of 7 S immunoglobulin or gamma globulin or a serumglobulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin for the manufacture of a medicament for thetreatment of choroidal neovascularisation, said active fraction havingthe ability to inhibit one or more activities selected from the groupconsisting of: a) choroidal neovascularisation, b) proliferation ofchoroidal endothelial cells, c) migration of choroidal endothelialcells, d) tube formation of choroidal endothelial cells, wherein saidactive fraction is present in the medicament in an amount effective toinhibit choroidal neovascularisation.
 88. Use of 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin for the manufacture of amedicament for the treatment of choroidal neovascularisation, said 7 Simmunoglobulin or gamma globulin or a serum globulin fraction that ismainly composed of IgG molecules or intravenous immunoglobulin havingthe ability to inhibit one or more activities selected from the groupconsisting of: a) choroidal neovascularisation, b) proliferation ofchoroidal endothelial cells, c) migration of choroidal endothelialcells, d) tube formation of choroidal endothelial cells, wherein said 7S immunoglobulin or gamma globulin or a serum globulin fraction that ismainly composed of IgG molecules or intravenous immunoglobulin ispresent in the medicament in an amount effective to inhibit theproliferation of choroidal endothelial cells.
 89. Use of an activefraction of 7 S immunoglobulin or gamma globulin or a serum globulinfraction that is mainly composed of IgG molecules or intravenousimmunoglobulin for the manufacture of a medicament for the treatment ofchoroidal neovascularisation, said active fraction having the ability toinhibit one or more activities selected from the group consisting of: a)choroidal neovascularisation, b) proliferation of choroidal endothelialcells, c) migration of choroidal endothelial cells, d) tube formation ofchoroidal endothelial cells, wherein said active fraction is present inthe medicament in an amount effective to inhibit the proliferation ofchoroidal endothelial cells.
 90. Use of 7 S immunoglobulin or gammaglobulin or a serum globulin fraction that is mainly composed of IgGmolecules or intravenous immunoglobulin for the manufacture of amedicament for the treatment of choroidal neovascularisation, said 7 Simmunoglobulin or gamma globulin or a serum globulin fraction that ismainly composed of IgG molecules or intravenous immunoglobulin havingthe ability to inhibit one or more activities selected from the groupconsisting of: a) choroidal neovascularisation, b) proliferation ofchoroidal endothelial cells, c) migration of choroidal endothelialcells, d) tube formation of choroidal endothelial cells, wherein said 7S immunoglobulin or gamma globulin or a serum globulin fraction that ismainly composed of IgG molecules or intravenous immunoglobulin ispresent in the medicament in an amount effective to inhibit themigration of choroidal endothelial cells.
 91. Use of an active fractionof 7 S immunoglobulin or gamma globulin or a serum globulin fractionthat is mainly composed of IgG molecules or intravenous immunoglobulinfor the manufacture of a medicament for the treatment of choroidalneovascularisation, said active fraction having the ability to inhibitone or more activities selected from the group consisting of: a)choroidal neovascularisation, b) proliferation of choroidal endothelialcells, c) migration of choroidal endothelial cells, d) tube formation ofchoroidal endothelial cells, wherein said active fraction is present inthe medicament in an amount effective to inhibit the migration ofchoroidal endothelial cells.
 92. Use of an active fraction of 7 Simmunoglobulin or gamma globulin or a serum globulin fraction that ismainly composed of IgG molecules or intravenous immunoglobulin for themanufacture of a medicament for the treatment of choroidalneovascularisation, wherein said active fraction being selected from thegroup consisting of IgG₁, IgG₂, IgG₃, IgG₄ and mixtures thereof.
 93. Theuse of claim 92, wherein said active fraction is composed of fragmentsof gamma immunoglobulin selected from the group consisting of F(ab′)₂,Fab′, Fab and Fc.
 94. Use of an active fraction of 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin for the manufacture of amedicament for the treatment of choroidal neovascularisation, saidactive fraction having the ability to inhibit one or more activitiesselected from the group consisting of: a) choroidal neovascularisation,b) proliferation of choroidal endothelial cells, c) migration ofchoroidal endothelial cells, d) tube formation of choroidal endothelialcells, wherein said active fraction is selected from the groupconsisting of IgG₁, IgG₂, IgG₃, IgG₄ and mixtures thereof.2
 95. The useof claim 94, wherein said active fraction is composed of fragments ofgamma immunoglobulin selected from the group consisting of F(ab′)₂,Fab′, Fab and Fc.
 96. Use of an active fraction of 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin for the manufacture of amedicament for the treatment of choroidal neovascularisation in anamount effective to inhibit choroidal neovascularisation and whereinsaid active fraction is selected from the group consisting of IgG₁,IgG₂, IgG₃, IgG₄ and mixtures thereof.
 97. The use of claim 96, whereinsaid active fraction is composed of fragments of gamma immunoglobulinselected from the group consisting of F(ab′)₂, Fab′, Fab and Fc.
 98. Useof an active fraction of 7 S immunoglobulin or gamma globulin or a serumglobulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin for the manufacture of a medicament for thetreatment of choroidal neovascularisation, said active fraction havingthe ability to inhibit one or more activities selected from the groupconsisting of: a) choroidal neovascularisation, b) proliferation ofchoroidal endothelial cells, c) migration of choroidal endothelialcells, d) tube formation of choroidal endothelial cells, wherein saidactive fraction is present in the medicament in an amount effective toinhibit choroidal neovascularisation and wherein said active fraction isselected from the group consisting of IgG₁, IgG₂, IgG₃, IgG₄ andmixtures thereof.
 99. The use of claim 98, wherein said active fractionis composed of fragments of gamma immunoglobulin selected from the groupconsisting of F(ab′)₂, Fab′, Fab and Fc.
 100. A method for the treatmentof choroidal neovascularisation which comprises administering to thepatient a preparation of 7 S immunoglobulin or gamma globulin or a serumglobulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin.
 101. A method for the treatment of choroidalneovascularisation which comprises administering to the patient apreparation of an active fraction of 7 S immunoglobulin or gammaglobulin or a serum globulin fraction that is mainly composed of IgGmolecules or intravenous immunoglobulin.
 102. A method for the treatmentof choroidal neovascularisation which comprises administering to thepatient a preparation of 7 S immunoglobulin or gamma globulin or a serumglobulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin in an amount effective to inhibit choroidalneovascularisation.
 103. A method for the treatment of choroidalneovascularisation which comprises administering to the patient apreparation of an active fraction of 7 S immunoglobulin or gammaglobulin or a serum globulin fraction that is mainly composed of IgGmolecules or intravenous immunoglobulin in an amount effective toinhibit choroidal neovascularisation.
 104. A method for the treatment ofchoroidal neovascularisation which comprises administering to thepatient a preparation of 7 S immunoglobulin or gamma globulin or a serumglobulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin, said 7 S immunoglobulin or gamma globulin ora serum globulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin having the ability to inhibit one or moreactivities selected from the group consisting of: a) choroidalneovascularisation, b) proliferation of choroidal endothelial cells, c)migration of choroidal endothelial cells, d) tube formation of choroidalendothelial cells.
 105. A method for the treatment of choroidalneovascularisation which comprises administering to the patient apreparation of an active fraction of 7 S immunoglobulin or gammaglobulin or a serum globulin fraction that is mainly composed of IgGmolecules or intravenous immunoglobulin, said active fraction having theability to inhibit one or more activities selected from the groupconsisting of: a) choroidal neovascularisation, b) proliferation ofchoroidal endothelial cells, c) migration of choroidal endothelialcells, d) tube formation of choroidal endothelial cells.
 106. A methodfor the treatment of choroidal neovascularisation which comprisesadministering to the patient a preparation of 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin, said 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin having the ability toinhibit one or more activities selected from the group consisting of: a)choroidal neovascularisation, b) proliferation of choroidal endothelialcells, c) migration of choroidal endothelial cells, d) tube formation ofchoroidal endothelial cells, wherein said 7 S immunoglobulin or gammaglobulin or a serum globulin fraction that is mainly composed of IgGmolecules or intravenous immunoglobulin is present in the medicament inan amount effective to inhibit choroidal neovascularisation.
 107. Amethod for the treatment of choroidal neovascularisation which comprisesadministering to the patient a preparation of an active fraction of 7 Simmunoglobulin or gamma globulin or a serum globulin fraction that ismainly composed of IgG molecules or intravenous immunoglobulin, saidactive fraction having the ability to inhibit one or more activitiesselected from the group consisting of: a) choroidal neovascularisation,b) proliferation of choroidal endothelial cells, c) migration ofchoroidal endothelial cells, d) tube formation of choroidal endothelialcells, wherein said active fraction is present in the medicament in anamount effective to inhibit choroidal neovascularisation.
 108. A methodfor the treatment of choroidal neovascularisation which comprisesadministering to the patient a preparation of 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin, said 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin having the ability toinhibit one or more activities selected from the group consisting of: a)choroidal neovascularisation, b) proliferation of choroidal endothelialcells, c) migration of choroidal endothelial cells, d) tube formation ofchoroidal endothelial cells, wherein said 7 S immunoglobulin or gammaglobulin or a serum globulin fraction that is mainly composed of IgGmolecules or intravenous immunoglobulin is present in the medicament inan amount effective to inhibit the proliferation of choroidalendothelial cells.
 109. A method for the treatment of choroidalneovascularisation which comprises administering to the patient apreparation of an active fraction of 7 S immunoglobulin or gammaglobulin or a serum globulin fraction that is mainly composed of IgGmolecules or intravenous immunoglobulin, said active fraction having theability to inhibit one or more activities selected from the groupconsisting of: a) choroidal neovascularisation, b) proliferation ofchoroidal endothelial cells, c) migration of choroidal endothelialcells, d) tube formation of choroidal endothelial cells, wherein saidactive fraction is present in the medicament in an amount effective toinhibit the proliferation of choroidal endothelial cells.
 110. A methodfor the treatment of choroidal neovascularisation which comprisesadministering to the patient a preparation of 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin, said 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin having the ability toinhibit one or more activities selected from the group consisting of: a)choroidal neovascularisation, b) proliferation of choroidal endothelialcells, c) migration of choroidal endothelial cells, d) tube formation ofchoroidal endothelial cells, wherein said 7 S immunoglobulin or gammaglobulin or a serum globulin fraction that is mainly composed of IgGmolecules or intravenous immunoglobulin is present in the medicament inan amount effective to inhibit the migration of choroidal endothelialcells.
 111. A method for the treatment of choroidal neovascularisationwhich comprises administering to the patient a preparation of an activefraction of 7 S immunoglobulin or gamma globulin or a serum globulinfraction that is mainly composed of IgG molecules or intravenousimmunoglobulin, said active fraction having the ability to inhibit oneor more activities selected from the group consisting of: a) choroidalneovascularisation, b) proliferation of choroidal endothelial cells, c)migration of choroidal endothelial cells, d) tube formation of choroidalendothelial cells, wherein said active fraction is present in themedicament in an amount effective to inhibit the migration of choroidalendothelial cells.
 112. A method for the treatment of choroidalneovascularisation which comprises administering to the patient apreparation of an active fraction of 7 S immunoglobulin or gammaglobulin or a serum globulin fraction that is mainly composed of IgGmolecules or intravenous immunoglobulin, wherein said active fractionbeing selected from the group consisting of IgG₁, IgG₂, IgG₃, IgG₄ andmixtures thereof.
 113. The method of claim 112, wherein said activefraction is composed of fragments of gamma immunoglobulin selected fromthe group consisting of F(ab′)₂, Fab′, Fab and Fc.
 114. A method for thetreatment of choroidal neovascularisation which comprises administeringto the patient a preparation of an active fraction of 7 S immunoglobulinor gamma globulin or a serum globulin fraction that is mainly composedof IgG molecules or intravenous immunoglobulin, said active fractionhaving the ability to inhibit one or more activities selected from thegroup consisting of: a) choroidal neovascularisation, b) proliferationof choroidal endothelial cells, c) migration of choroidal endothelialcells, d) tube formation of choroidal endothelial cells, wherein saidactive fraction is selected from the group consisting of IgG₁, IgG₂,IgG₃, IgG₄ and mixtures thereof.
 115. The method of claim 35, whereinsaid active fraction is composed of fragments of gamma immunoglobulinselected from the group consisting of F(ab′)₂, Fab′, Fab and Fc.
 116. Amethod for the treatment of choroidal neovascularisation which comprisesadministering to the patient a preparation of an active fraction of 7 Simmunoglobulin or gamma globulin or a serum globulin fraction that ismainly composed of IgG molecules or intravenous immunoglobulin in anamount effective to inhibit choroidal neovascularisation and whereinsaid active fraction is selected from the group consisting of IgG₁,IgG₂, IgG₃, IgG₄ and mixtures thereof.
 117. The method of claim 116,wherein said active fraction is composed of fragments of gammaimmunoglobulin selected from the group consisting of F(ab′)₂, Fab′, Faband Fc.
 118. A method for the treatment of choroidal neovascularisationwhich comprises administering to the patient a preparation of an activefraction of 7 S immunoglobulin or gamma globulin or a serum globulinfraction that is mainly composed of IgG molecules or intravenousimmunoglobulin, said active fraction having the ability to inhibit oneor more activities selected from the group consisting of: a) choroidalneovascularisation, b) proliferation of choroidal endothelial cells, c)migration of choroidal endothelial cells, d) tube formation of choroidalendothelial cells, wherein active fraction is present in the medicamentin an amount effective to inhibit choroidal neovascularisation andwherein said active fraction is selected from the group consisting ofIgG₁, IgG₂, IgG₃, IgG₄ and mixtures thereof.
 119. The method of claim118, wherein said active fraction is composed of fragments of gammaimmunoglobulin selected from the group consisting of F(ab′)₂, Fab′, Faband Fc.
 120. Pharmaceutical composition essentially consisting of anactive fraction of 7 S immunoglobulin or gamma globulin or a serumglobulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin as pharmaceutically active compound. 121.Pharmaceutical composition essentially consisting of an active fractionof 7 S immunoglobulin or gamma globulin or a serum globulin fractionthat is mainly composed of IgG molecules or intravenous immunoglobulinas pharmaceutically active compound in an amount effective to inhibitchoroidal neovascularisation.
 122. Pharmaceutical compositionessentially consisting of an active fraction of 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin as pharmaceutically activecompound, said active fraction having the ability to inhibit one or moreactivities selected from the group consisting of: a) choroidalneovascularisation, b) proliferation of choroidal endothelial cells, c)migration of choroidal endothelial cells, d) tube formation of choroidalendothelial cells.
 123. Pharmaceutical composition essentiallyconsisting of an active fraction of 7 S immunoglobulin or gamma globulinor a serum globulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin as pharmaceutically active compound, saidactive fraction having the ability to inhibit one or more activitiesselected from the group consisting of: a) choroidal neovascularisation,b) proliferation of choroidal endothelial cells, c) migration ofchoroidal endothelial cells, d) tube formation of choroidal endothelialcells. wherein said active fraction is present in the medicament in anamount effective to inhibit choroidal neovascularisation. 124.Pharmaceutical composition essentially consisting of an active fractionof 7 S immunoglobulin or gamma globulin or a serum globulin fractionthat is mainly composed of IgG molecules or intravenous immunoglobulinfor the manufacture of a medicament for the treatment of choroidalneovascularisation, said active fraction having the ability to inhibitone or more activities selected from the group consisting of: a)choroidal neovascularisation, b) proliferation of choroidal endothelialcells, c) migration of choroidal endothelial cells, d) tube formation ofchoroidal endothelial cells, wherein said active fraction is present inthe medicament in an amount effective to inhibit the proliferation ofchoroidal endothelial cells.
 125. Pharmaceutical composition essentiallyconsisting of an active fraction of 7 S immunoglobulin or gamma globulinor a serum globulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin as pharmaceutically active compound, saidactive fraction having the ability to inhibit one or more activitiesselected from the group consisting of: a) choroidal neovascularisation,b) proliferation of choroidal endothelial cells, c) migration ofchoroidal endothelial cells, d) tube formation of choroidal endothelialcells, wherein said active fraction is present in the medicament in anamount effective to inhibit the migration of choroidal endothelialcells.
 126. Pharmaceutical composition essentially consisting of anactive fraction of 7 S immunoglobulin or gamma globulin or a serumglobulin fraction that is mainly composed of IgG molecules orintravenous immunoglobulin as pharmaceutically active compound, whereinsaid active fraction is one, two or three of the components selectedfrom the group consisting of IgG₁, IgG₂, IgG₃, IgG₄.
 127. Thepharmaceutical composition of claim 126, wherein said active fraction iscomposed of fragments of gamma immunoglobulin selected from the groupconsisting of F(ab′)₂, Fab′, Fab and Fc.
 128. Pharmaceutical compositionessentially consisting of an active fraction of 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin as pharmaceutically activecompound in an amount effective to inhibit choroidal neovascularisationand wherein said active fraction is one, two or three of the componentsselected from the group consisting of IgG₁, IgG₂, IgG₃, IgG₄.
 129. Thepharmaceutical composition of claim 128, wherein said active fraction iscomposed of fragments of gamma immunoglobulin selected from the groupconsisting of F(ab′)₂, Fab′, Fab and Fc.
 130. Pharmaceutical compositionessentially consisting of an active fraction of 7 S immunoglobulin orgamma globulin or a serum globulin fraction that is mainly composed ofIgG molecules or intravenous immunoglobulin as pharmaceutically activecompound, said active fraction having the ability to inhibit one or moreactivities selected from the group consisting of: a) choroidalneovascularisation, b) proliferation of choroidal endothelial cells, c)migration of choroidal endothelial cells, d) tube formation of choroidalendothelial cells, wherein said active fraction is one, two or three ofthe components selected from the group consisting of IgG₁, IgG₂, IgG₃,IgG₄.
 131. The pharmaceutical composition of claim 130, wherein saidactive fraction is composed of fragments of gamma immunoglobulinselected from the group consisting of F(ab′)₂, Fab′, Fab and Fc.